Sinha et al_BIOREMEDIATION OF CONTAMINATED SITES.pdf

In: Solid Waste Management and Environmental Remediation ISBN: 978-1-60741-761-3 Editor: Timo Faerber and Johann Herzog 2009 Nova Science Publishers, Inc.

Chapter 1

BIOREMEDIATION OF CONTAMINATED SITES: A LOW-COST NATURES BIOTECHNOLOGY FOR ENVIRONMENTAL CLEAN UP BY VERSATILE

MICROBES, PLANTS & EARTHWORMS

Rajiv K. Sinha1, Dalsukh Valani 2 , Shanu Sinha 3 Shweta Singh4 & Sunil Herat5

1 School of Engineering (Environment), Griffith University, Nathan, 2 Master of Engineering (Environment) (Applied for Ph.D), Griffith University

3 Research & Development (Biotechnology), Wesley Hospital, Brisbane 4 Ph.D (Botany), University of Rajasthan, India

5 School of Engineering (Environment), Griffith University

Keywords: Phytoextraction; Phytovolatalization; Phytostabilization; Phytostimulation; Rhizofiltration; Phytotransformation; Phytodegradation; Excluder Plants; Hyper-accumulator Plants Species; Transgenic (Genetically Engineered) Plants Combating Soil Pollution; Symbiotic Engineering.

Biotransformation; Microbial Detoxification of Metals; Microbial Destruction of Toxic Organics & Hazardous Wastes; Plant Assisted Microbial Destruction of Toxic Compounds;

Earthworms Bio-accumulate & Bio-degrade & Bio-transform Toxic Chemicals; Earthworms Immobilize Soil Contaminants;

1. INTRODUCTION Large tract of arable land is being chemically contaminated due to mining activities,

heavy use of agro-chemicals in farmlands, landfill disposal of toxic wastes and other developmental activities like oil and gas drilling. No farmland of world especially in the developing nations are free of toxic pesticides, mainly aldrin, chlordane, dieldrin, endrin, heptachlor, mirex and toxaphene. According to National Environment Protection Council

Corresponding Author: Campus, Brisbane, QLD-4111, Australia

Rajiv K. Sinha, Dalsukh Valani, Shanu Sinha, et al. 2

there are over 80,000 contaminated sites in Australia- 30,000 in NSW, another 30,000 in Queensland, 10,000 in Victoria, 4000 in South Australia, another 4000 in Western Australia, 1000 in Northern Territory and 500 each in Tasmania and the Australian Capital Territory (ACT). There are 40,000 contaminated sites in US; 55,000 in just six European countries and 7,800 in New Zealand. There are about 3 million contaminated sites in the Asia-Pacific. These also include the abandoned mine sites along with the closed landfills. The contaminated sites mostly contain heavy metals cadmium (Cd), lead (Pb), mercury (Hg), zinc (Zn) etc. and chlorinated compounds like the PCBs and DDT. (UNEP, 1996; Eshwaran et al. 2001). Cleaning them up mechanically by excavating the huge mass of contaminated soils and disposing them in secured landfills will require billions of dollars. There is also great risk of their leaching underground (aggravated by heavy rains) and contaminating the groundwater. Contaminated soils and waters pose major environmental, agricultural and human health problems worldwide.

Traditionally, remediation of chemically contaminated soils involves off-site

management by excavating and subsequent disposal by burial in secured landfills. This method of remediation is very costly affair and merely shifts the contamination problem elsewhere. Additionally, this involves great risk of environmental hazard while the contaminated soils are being transported and migration of contaminants from landfills into adjacent lands and water bodies by leaching. Soil washing for removing inorganic contaminants from soil is another alternative to landfill burial, but this technique produce a residue with very high metal contents which requires further treatment or burial.

Since the late 1980s, after the chemical and mechanical treatments of lands and water bodies and thermal treatment (incineration) of hazardous wastes proved economically and environmentally unsustainable, focus shifted towards the biological methods which are cost-effective as well as environmentally sustainable and also socially acceptable. Bioremediation is a soft bioengineering technique to clean up contaminated lands/sites using microbes (bacteria or fungus), plants (terrestrial and aquatic) and earthworms. It is also a technique to stabilize the eroded lands and prevent soil erosion. Bioremediation works carried out by the microorganisms are called micro-remediation while those performed by plants are called phyto-remediation. Earthworms have also been found to perform some environmental cleaning jobs and is termed as vermi-remediation.

I. MICROBES ASSISTED BIOREMEDIATION (MICROREMEDIATION) Microbes are adapted to thrive in adverse conditions of high acidity/alkalinity/toxicity

and high temperature. They can develop biological resistance against any toxic substance in the environment due to special jumping genes. Hence while a number of them may be killed due to high toxicity, some resistant microbes survive and are cultured for further use. Under favorable conditions of growth e.g. pH, temperature and moisture and adequate supply of nutrients like vitamins, magnesium, manganese, copper, sulfur, potassium, phosphorus and nitrogen, microbes can biodegrade/biotransform the complex hazardous organic chemicals into simpler and harmless ones. After the use of super bug in cleaning up oil spills, there has been several successful stories of microbial technique in clean-up of contaminated lands and

Bioremediation of Contaminated Sites: A Low-Cost Natures Biotechnology 3

soils. (USGS, 1997). The Microbiological Resource Centers (MIRCENS) at Cairo, Egypt is examining the use of microbes in degrading persistent pesticides pollutants. (UNEP Reports, 1996-2006).

Whilst microbial remediation (bioremediation) is a well established technology for the removal of organic soil contaminants, the use of microorganisms to transform inorganic contaminants like heavy metals is still being investigated. Environmentalists are viewing microbes as an eco-friendly nano-factories for metal remediation through biotechnological applications employing microbes, such as yeast, bacteria, algae, diatoms and actinomycetes.

There are bacteria which can also ingest the most toxic cyanide from water.

Microbial Bioremediation of Heavy Metals Metals play important role in the life processes of microbes. Some metals such as

chromium (Cr), calcium (Ca), magnesium (Mg), manganese (Mn), copper (Cu), sodium (Na), nickel (Ni) and zinc (Zn) are essential as micronutrients for various metabolic functions and for redox functions. Other metals have no biological role e.g. cadmium (Cd), lead (Pb), mercury (Hg), aluminum (Al), gold (Au) and silver (Ag). They are non-essential and potentially toxic to soil microbes. Some of them e.g. Cd2+, Ag2+, Hg2+ tend to bind the SH groups of enzymes and inhibit their activity (Turpeinen, 2002).

Soil contamination by heavy metals may repress or even kill parts of the microbial community in soil. Interaction of metals with cellular proteins/enzymes are more commonly implicated in causing toxicity than interaction with membranes. Binding affects the structure and function of proteins and enzymes.

Metal Tolerance & Resistance by Microbes It is generally assumed that the exposure to metals leads to the establishment of a

tolerant/resistant microbial population. Microbial resistance is defined as the ability of a micro-organism to survive toxic effects of metal exposure by means of a detoxification mechanism produced in direct response to the heavy metal species concerned. Microbial tolerance is defined as the ability of a micro-organism to survive metal toxicity by means of intrinsic properties and or environmental modification of toxicity.

Soil micro-organisms have been shown to bio-accumulate metals in tissues in concentrations up to 50 times higher than the surrounding soil.

Micro-organisms employ a variety of mechanisms to resist and cope with toxic metals. The principal mechanism of resistance of inorganic metals by microbes are metal oxidation, metal reduction, methylation, demethylation, enzymatic reduction, metal-organic complexion, metal ligand degradation, intracellular and extracellular metal sequestration, metal efflux pumps, exclusion by permeability barrier and production of metal chelators such as metallothioneins and biosurfactants.

The Process and Mechanism of Microbial Remediation


Microbial remediation of toxic metals occurs in two ways 1). Direct reduction by the activity of the bacterial enzyme metal reductase. It is

applied for groundwater decontamination, using bioreactors (pump & treat) and also for soils after excavation (pulping or heaping and inoculation with appropriate microbial consortium). These techniques are ex-situ methods, and very expensive and has low metal extraction efficiencies.

2). Indirect reduction by biologically produced hydrogen sulfide (H2S) by sulfate reducing bacteria to reduce and precipitate the metals. This is an in-situ method, and an environmentally sound & inexpensive alternative to pump & treat (for contaminated groundwater) or excavate & treat (for contaminated soils). Microbial growth is induced in sub-surface zones by injecting substrates. The migrating metals are intercepted and immobilized by precipitation with biologically produced H2S.

There are at least three major microbial processes that influence the bioremediation of

metals and these are

1). Biosorption & Bioaccumulation Biosorption is sequestration of the positively charged heavy metal ions (cations) to the

negatively charged microbial cell membranes and polysachharides secreted in most of the bacteria on the outer surfaces through slime and capsule formation. From the surface the metals are transported into the cell cytoplasm through the cell membrane with the aid of transporter proteins and get bioaccumulated.

2). Biologically Catalysed Immobilization

Inside the microbial cells, metal ions gets fixed to Iron (Fe)-Oxides and into organic colloids and becomes immobilised. This is achieved by enzymatic reduction by microbes (described below).

3). Biologically Catalysed Solubilization

Metal reducing bacteria enzymatically reduce and also under appropriate conditions, solubilize oxide minerals. Such dissolution reaction have been shown to release cadmium (Cd), nickel (Ni) and zinc (Zn) into solution during reduction of goethite (a form of Fe-oxide) by anaerobic bacterium Closteridium spp.

Microorganisms do not actually biodegrade inorganic metals, but changes (bio-transform) their oxidation state. This can lead to an increase in solubility (and subsequent removal by leaching), or precipitation and reduction in bioavailability. Metallic residues (heavy metals) may be converted into metal-organic combinations that have less bioavailability (to pass into human food chain) than the metal-mineral combinations of the heavy metals. Microbes transform the oxidation states of several toxic metals and increase their bioavailability in the rhizosphere (root zone) thus facilitating their absorption and removal by hyper-accumulating plants by phytoremediation.

Many divalent metal cations like Mn2+, Fe2+ and Zn2+ are very similar in structure. Also, the structure of oxyanions, such as chromate, resembles that of sulphate. Evolution has endowed micro-organisms with effective mechanisms to distinguish between similar metal


ions and between toxic and non-toxic metals. Microbes have solved this problem by developing two types of uptake mechanisms and systems for metal ions -

1). Selective, substrate-specific uptake system that are slow and require considerable cellular energy (ATP) and is only produced by the cell in times of need;

2). Substrate-non-specific rapid system, that transport metals using a chemiosmotic gradient across the cytoplasmic membrane of the bacteria rather than using ATP. (Nies, 1999).

Even highly evolved, substrate-specific uptake mechanisms may not prevent entry of

toxic metals in cells. Once inside, metal cations can interact with various cellular components including cell membranes, proteins and nucleic acids. Incompletely filled d-orbitals allow metals to form complex compounds with organic ligands, such as the proteins, nucleic acids & cell wall materials of micro-organisms. The ability of microbial cell surfaces to form complex with metals lies in their net negative charge at normal growth pH. The outer membrane of Gram negative bacteria effectively complexes metals including magnesium (Mg), nickel (Ni), strontium (Sr), manganese (Mn), lead (Pb), iron (Fe), sodium (Na) and calcium (Ca). In Gram eve bacteria the, the net eve charge results from the phosphates and carboxyl groups of lipopolysaccharide molecules, while the eve charge in Gram positive bacteria results largely from teichoic acid. A more negative cell surface charge may more effectively attract and bind toxic metal cations. Toxic metals readily binds to sulfhydryl group of proteins. (Nies, 1999; Sandrin & Hoffman, 2006).

The resemblance of some toxic heavy metals to essential metabolite (minerals) allows them to readily enter into the microbial cells. Thus, chromate (Cr) is often mistakenly taken up in place of sulphate (S), arsenate (As) is mistaken for phosphate (P), cadmium (Cd) is used as an enzyme co-factor instead of zinc (Zn) or calcium (Ca), nickel (Ni) and cobalt (Co) is mistaken for iron (Fe), and zinc (Zn) is very commonly mistaken for magnesium (Mg). (Nies, 1999; Sandrin & Hoffman, 2006).

MICROBIAL DETOXIFICATION OF METALS One or more of the resistance mechanisms allows microbes to function in metal

contaminated environments and detoxify them. Micro-organisms can detoxify metals by valence transformation, by extracellular chemical precipitation or by volatilization.

Generally, microbial transformations & detoxifications of metals occur either by redox conversions (reduction) of inorganic forms or conversions from inorganic to organic forms and vice versa. Most toxic heavy metals are less soluble and less toxic when in reduced state than in oxidised state. Reduction of metals can occur through dissimilatory metal reduction, where microbes utilize metals as terminal electron acceptor for anaerobic respiration.

To date arsenic (As), chromium (Cr), mercury (Hg), uranium (U) and selenium (Se) have responded well to detoxification by microbial reduction. Oscillatoria spp. (a blue-green algae), Chlorella vulgaris & Chlamydomonas spp. (green algae), Arthrobacter, Agrobacter, Enterobacter & Pseudomonas aeruginosa are some metal reducing microbes. (Ramasamy et. al., 2006).


1). Microbial reduction of chromium (vi) to cr (iii) Chromium is widely used in many industrial & developmental activities, such as in

leather & tannery industries, electroplating, steel and automobile manufacturing, production of paint pigments and dyes, refractory and in wood preservation. Its world production is in the order of 10,000,000 tons per year. It is a hazardous contaminant and is a serious threat to human health as it readily spreads beyond the site of initial contamination through aquatic ecosystems and groundwater. (Viti & Giovannetti, 2006).

Chromium in environment is able to exist in several oxidation states, ranging from Cr (II) to Cr (VI), but in soils the most stable & common forms are trivalent Cr (III) and hexavalent Cr (VI) species. The trivalent & hexavalent forms can inter-convert. Cr (III) is essential for animal and human nutrition. Utmost consideration is given to Cr (VI) because it is water-soluble, highly toxic and mutagenic to most organisms and carcinogenic for humans. It is also involved in causing birth defects and the decrease of reproductive health.

A wide range of microbes have been found to have chromium (Cr) tolerance, resistance and reducing ability. The blue-green algae Nostoc have been reported to exist in a soil chronically polluted by chromium (about 5000 mg/kg of soil) from leather tannery. Other microbes tolerating / resisting Cr (IV) levels are Arthrobacter crystallopoites (500 mg/L), Pseudomonas spp. CRB 5 (520 mg/L), Bacillus maroccanus ChrA21 (1040 mg/L), Corynebacterium hoagii Chr B20 (1144 mg/L), Bacillus cereus ES04 (1500 mg/L). (Viti & Giovannetti, 2006). Anaerobic sulfate reducing and methanogenic bacteria possess inherent abilities to sorb more than 90 % of chromium to its cell biomass. Microbe reduce the highly soluble chromate ions to Cr (III), which under appropriate conditions precipitates as Cr(OH)3. Organic matter (carbon sources) of the soil plays an important role in the reduction of Cr (VI) to Cr (III) by creating reducing conditions, such as increasing activities of soil microbes and by acting as an electron donor,& also by indirectly lowering the oxygen level of the soil (due to increased microbial respiration). Carbon sources, such as organic acids, manure, mollases, have been proposed to improve Cr (VI) reduction, that otherwise is very slow.

2). Microbial reduction of uranium (vi) to u (iv)

Microbe reduce uranyl carbonate, which is exceedingly soluble in carbonate-bearing groundwater, to highly insoluble U (IV) which precipitates from solution as the uranium oxide mineral uraninite. Recently scientists have succeeded in microbial binding of U (IV), which is then converted by the living cell to U (IV) and precipitated intracellularly. The Geobacter spp. have been found to work well to remove uranium (U) from groundwater. (Anderson et. al., 2003).

3). Microbial reduction of selenium (vi) to elemental se (0)

It causes precipitation of the selenium metal and reduced bioavailability. In addition, SeO4 can be microbially methylated to volatile dimethyl selenide which escape from soil.

Role of Iron & Sulfate Reducing Bacteria in Reducing Toxic Metals The iron (Fe) reducing bacterium e.g. Geobacter spp. and the sulfur reducing bacterium

e.g. Desulfuromonas spp. are also capable of reducing the toxic metals. They have been found


to colonise habitats with elevated metal concentrations. G. metallireducens, a strict anaerobe is able to reduce manganese (Mn), such as toxic Mn (IV) to Mn (II), and reduce uranium (U), such as toxic U (VI) to U (IV). The G. sulfurreducens & G. metallireducens are able to reduce chromium (Cr) such as highly toxic Cr (VI) to harmless Cr (III). The sulfur reducing bacterium is strictly anaerobic and gram-negative. It acquires its energy from sulfur (S) respiration and completely oxidises acetic acid (organic acid) with sulfur (S) to carbon dioxide (CO2). Reduction of sulfur (S) produces hydrogen sulfide (H2S) which reacts with heavy metal ions to form less toxic insoluble metal-sulfides. Furthermore, these bacteria are also able to enzymatically reduce and precipitate these heavy metals. (Bruschi and Goulhen, 2006). This approach of indirect metal reduction by biologically produced H2S by sulfur reducing bacteria was developed in the 1980s and has been used commercially up to industrial scale. They lead to selective metal precipitation, such as copper (Cu) and zinc (Zn) sulfates and acidity removal.

Some Success Stories 1). Pilot plants developed by Shell Research Ltd. and Budelco BV, France, using an

undefined consortium of SRB (sulfate reducing bacteria), have been used successfully to remove zinc (Zn) and sulfate. The metals were precipitated as sulfides. Acetic acid, produced by the SRB was removed by the methanogenic bacteria present in the consortium. This technology has been scaled up and now capable of treating 7000 cubic meter (cum) of contaminated soil per day. (Bruschi and Goulhen, 2006)

2). In-situ bioremediation of uranium contaminated sites have been conducted successfully with Desulfosphorosinus spp. and Closteridium spp. (Bruschi & Goulhen, 2006).

3). Treatment of other metals using anaerobic bioreactor with SRB community culture has been reported for phosphogypsum, a hazardous waste from fertilizer industries and for lead wastes from car batteries. Lead waste is reduced to less hazardous PbS (Galena).

Microbial Methylation of Metals : Another Mechanism of Detoxification Microbial methylation also plays an important role in metal detoxification, because

methylated compounds are often volatile. Mercury, Hg (II) can be biomethylated by a number of bacteria e.g. Pseudomonas spp., Bacillus spp., Closteridium spp. and Escherichia spp. to gaseous methyl mercury. Biomethylation of arsenic (As) to gaseous arsines, selenium (Se) to volatile dimethyle selenide, and lead (Pb) to dimethyl lead has been observed in various contaminated soils. (Ramasamy et, al., 2006).

Acidophilic iron and sulfur oxidizing bacteria are able to leach high concentrations of arsenic (As), cadmium (Cd), copper (Cu), cobalt (Co) and zinc (Zn) from contaminated soils. Also heavy metals can be precipitated as insoluble sulfides indirectly by the metabolic activity of sulfate reducing bacteria. (Ramasamy et, al., 2006).


Table 1. Naturally Occurring Bacteria Capable of Destroying Some Hazardous Wastes and Chemicals by Biodegradation

Organisms Chemicals Degraded Flavobacterium spp. Organophosphates Cunniughamela elegans & Candida tropicalis

PCBs (Polychlorinated Biphenyls) & PAHs (Polycyclic Aromatic Hydrocarbons);

Alcaligenes spp. & Pseudomonas spp.

PCBs, halogenated hydrocarbons and alkylbenzene sulphonates, PCBs, organophosphates, benzene, anthracene, phenolic compounds, 2,4 D, DDT and 2,4,5-trichlorophenoxyacetic acid etc.

Actinomycetes Raw rubber Nocardia tartaricans Chemical Detergents (Ethylbenzene) Closteridium Lindane Arthrobacter & Bacillus Endrin Trichoderma & Pseudomonas

Malathion

Sources: Various Publications of UNEP, WWF and WHO (1992-2002)

4. MICROBIAL DESTRUCTION OF TOXIC ORGANICS Several microbes have been identified in nature which can break down the hazardous

organic substances in the environment (soil & water) including the xenobiotic compounds such as pesticides, polycyclic aromatic hydrocarbons (PAHs) and the chlorinated substances like polychlorinated biphenyls (PCBs) in due course of time. Majority of the organochlorines appears to be biotransformed, forming cunjugates with the soil humic matter. Scientists at IIT, Madras have discovered a bacterial mixture which breaks down the deadly pesticide endosulphan into simple inorganic chemicals under both aerobic and anaerobic conditions.

1). Bacterial degradation of toxic organics

Naturally occurring aerobic bacteria have been found to decompose both natural and the synthetic hazardous organic materials to harmless CO2 and water. However, anaerobic microbes are important for degrading the halogens (reductive dehalogenation) and nitrosamine, reduction of epoxides to olefins, reduction of nitro groups and ring fission of aromatic structures.

2). Fungal degradation of toxic organics

All fungus, but more specifically the wood-decaying members of Basidiomycetes called the white rot fungus (WRF) are most efficient microbe that de-polymerize and even mineralize the lignin contents which is hard to be degraded. The fungus produce a powerful extra-cellular ligninolytic enzyme system which is a strong oxidant. Three main types of oxido-reductase activities can be found in this enzymatic system the polyphenoloxidases, peroxidases and auxiliary H2O2 generating oxidases. More specifically, the key lignolytic enzymes synthesized by WRF are laccase(polyphenoloxidase). Both, whole fungal cultures of WRF as well as the lignin-degrading enzymes were found to be useful to the


bioremediation of a wide number of toxic pollutants including the recalcitrant xenobiotics and more importantly the textile dyes which is highly resistant to washing, chemical solvents, action of sunlight and microbial attack. Rate of degradation is affected by atmospheric oxygen concentration, nutrient nitrogen and the source of carbon.

Fungal Destruction of Chlorinated Aromatic Compounds A very promising fungus which has the arsenal to degrade and destroy an assortment of

persistent chlorinated aromatic compounds have been identified. It is the white-rot fungus (Phanerochaete chrysosporidium). In Turkey, scientists have found that P. chrysosporidium can biodegrade LDPE plastic bags containing 12 % starch. Scientists in Japan have used biochemical machining and enzymes found in filamentous fungi to biodegrade or cut down the molecular chains present in biodegradable plastics.

Use of Yeast for Removal of Textile Dyes from Polluted Waters Industrial textile dyes have been designed and synthesized to be highly resistant to

washing and action of chemical solvents and sunlight. There are currently more than 10,000 different textile dyes commercially available in the world markets. Azo, indigoid and anthraquinone are the major chromophores used in the textile industries. They are complexed with heavy metals copper (Cu), cobalt (Co) and chromium (Cr) and are of considerable public health concern. Conventional treatments like adsorbent by activated carbons is very expensive and do not remove heavy metals.

Living yeast biomass specifically the strains of Candida tropicalis isolated from sewage has been shown to bio-absorb various textile dyes the reactive black and red, and the remazol blue. Maximum bio-accumulation capacity range from 79 mg/g (per gram of yeast biomass) for reactive red to 112 mg/g for remazol blue. It has significantly lower operational costs and the heavy metals are also absorbed and removed. (Donmez, 2002).

Table 2. Naturally Occurring Fungus Capable of Destroying Some Hazardous Wastes

and Chemicals by Biodegradation

Organisms Chemicals Degraded Phanerochaete chrysoporium,

Halocarbons such as lindane, pentachlorophenol,

P. sordida & Trametes hirsute

DDT, DDE, PCBs, 4,5,6-trichlorophenol, 2,4,6-trichlorophenol, dichlorphenol, and chlordane

Zylerion xylestrix Pesticides / Herbicides (Aldrin, dieldrin, parathion and malathion)

Mucor Dieldrin Yeast (Saccharomyces) DDT



Table 3. Some Hazardous Organic Chemicals Degradable By Anaerobic Digestion

1. Acetylsalicylic acid 13. Dimethoxy benzoic acid 25. Nitrobenzene 2. Acetaldehyde 14. Dimethylphthalate 26. Pentanol 3. Acetic anhydride 15. Ethyl acetate 27. Phenol 4. Acetone 16. Ferulic acid 28. Phthalic acid 5. Benzoic acid 17. Formaldehyde 29. Polyethylene glycol 6. Benzyl alcohol 18. Glycerol 30. Propanal 7. Butanol 19. Hexanoic acid 31. Proponol 8. Butylene glycerol 20.Methanol 32. Pyrogallol 9. Butyric acid 21. Methyl acetate 33. Sorbic acid 10. Catechol 22. Methyl acrylate 34. Valeric acid 11. Crotonic acid 23. Methyl ethyl ketone 35. Vannilic acid 12. Diethylphthalate 24. Methyl formate 36. Vinyl acetate


Microbial Destruction of Polycyclic Aromatic Hydrocarbons (PAHs) Among the most abundant environmental pollutants, the aromatic compounds are of

major concern because of their persistence and toxicity. PAHs are obiquitous in nature found throughout the environment in air, water and soil. PAHs are group of over 100 chemicals of which anthracene, benzo (a) pyrene, naphthalene, pyrene are common. They are formed by the incomplete combustion of fossil fuels (coal & oil) and wood. They are emitted from petroleum refineries, coal and oil power plants, coking plants, asphalt production plants, aluminum plants, industrial machinery manufacture and paper mills. They readily evaporate in air. People may be exposed to PAHs in the soil where coal, wood or petrol has been burnt. Food grown on such soils may contain PAH. However, several PAHs breaks down in sunlight in air over a period of days to weeks. Soil microbes can degrade them in weeks to months.

US EPA has identified 16 PAHs as priority pollutants made of two or more fused benzene rings with potential for high bio-magnification, and therefore, toxic to living organisms. PAHs are able to bio-accumulate in plants and animals including human beings. Benzo (a) pyrene, benzo (a) anthracene and chrysene are carcinogenic to humans, mutagenic in animal and bacterial cells, and also teratogenic, causing genetic defect in humans. Some PAHs can also induce immunodepressive effects. These chemicals are thermodynamically stable, have very low aqueous solubility, are highly hydrophobic, and tend to be strongly associated with particles surface in the environment. Most of them are resistant to volatilization and photolysis and are therefore, very persistent under natural conditions with half-lives in soils ranging from 26 days for phenanthrene (a volatile and degradable compound) to 6,250 days for benzo (a) anthracene (a recalcitrant compound). (Sims & Overcash, 1983).

Bacteria capable of destroying PAHs are species of Pseudomonas, Alcaligenes, Rhodococcus, Sphingomonas and Mycobacterium. They utilize PAHs as their sole carbon and energy source. A diverse group of fungi, both ligininolytic and non-ligininolytic, are able to degrade PAHs. But they transform them co-metabolically to detoxified metabolites and do not use them as source of carbon and energy like bacteria. (Sutherland, 1992). Both


prokaryotic and eukaryotic algae have been found to degrade PAHs in aquatic environments. The blue-green algae (Cyanobacteria) oxidize PAHs under photoautotrophic conditions to form hydroxylated intermediates.

Mechanism of Microbial Action in Degradation of Toxic Compounds This is achieved by biodegradation and biotransformation of complex toxic chemicals

into harmless simpler biochemical products by heterotrophic microbes which cannot produce their own food and in order to survive extract growth nutrients from the host material (which may be an organic or inorganic waste) by decomposing it. Microbes can also transform toxic substances into energy source. Microbes grow geometrically and in the process of decomposition produce a huge biomass of new cells. Hence the biodegradation process become faster with time.

3-

1 2 2 3 3 4 4 5 6 2 7 2V (organic material) + V O + V NH + V PO (microbes) V (new cells) + V CO + V H O Oxygen (O2), ammonia (NH3), and phosphate (PO43-) are the nutrients needed by the

microbes to oxidize the organic matter in the waste. Biodegradation by microbes is system-specific. Unless a proper microbial consortium is

both developed and maintained, a compound may not degrade in that system. Microorganisms adapt to degrade new synthetic compounds either by utilizing catabolic enzymes they already possess or by acquiring new metabolic pathways. In any event the biological systems can lower the cost of downstream processes by reducing organic load. While a microbial community that is resistant to moderate levels of heavy metals can be developed, the accumulation of metal precipitates on the bacterial biomass may severely inhibit their activity. Hence, a pretreatment system to remove metal contaminants may be needed. If the toxin is non-biodegradable, microbial strains that are resistant to the toxins must be enriched.

Microorganisms break down the complex hydrocarbons in the hazardous waste by using the three general mechanisms- aerobic and anaerobic respiration and fermentation. Aerobic process requires adequate supply of oxygen and the biodegradation process is rapid and more complete and there is no problematic end products like methane and hydrogen sulfide.

(a). Anaerobic Degradation

It is a sequential, biologically destructive process in which the complex hydrocarbons of hazardous wastes are converted into simpler molecules of carbon dioxide and methane by anaerobic microbes. The anaerobes use oxygen that is combined chemically with other elements, such as nitrates, carbonates, or sulphates. If nitrates are source of oxygen, nitrogen is formed and if sulfates, hydrogen sulfide is formed under anaerobic process. They also require complete absence of free oxygen molecules. They require a narrow pH range than the aerobic systems. Anaerobic microbes are important for degrading the halogens (reductive dehalogenation) and nitrosamine, reduction of epoxides to olefins, reduction of nitro groups and ring fission of aromatic structures. Several microbes have been identified which can break down the chlorinated substances like PCBs. Majority of the organochlorines appears to be biotransformed, forming cunjugates with the soil humic matter.


Table 4. Some Organic Compounds Transformed through Anaerobic Process

Toxic Chemicals Biological Transformation to Harmless Products 2,4-Dinitrophenol Methane 2,4-Dimethyl phenol Methane Carbon tetrachloride Degrade slowly Aldrin Dieldrin DDT Dechlorinated to DDE Toxaphene Reduced and dechlorinated Halogenated Benzoates Degraded after dechlorination to CO2 and CH4 Benzene Degraded to CO2 and CH4 Isopropylbenzene Degraded to CO2 and CH4 Ethylbenzene Degraded to CO2 and CH4 Toluene Degraded to CO2 and CH4

Sources: Various Publications of UNEP, WWF and WHO (1992-2002) The microbiology of anaerobic digestion consists of two distinct groups of anaerobic

microorganisms- the acetogens and the methanogens present at several trophic levels. At the higher levels, organisms attack waste molecules through hydrolysis and fermentation, reducing them to simpler hydrocarbons methane (CH4) and carbon dioxide (CO2) in the last two steps of the destructive process. It is crucial that a delicate balance of population must be maintained between the two distinct groups of essential anaerobes- the acetogens and the methanogens in the final stages of reaction. The acetogens convert most of the hydrocarbons of waste to acetate, CO2, and H2 and some organic acids. The methanogens convert organic acids and CO2 to CH4 in the presence of the available H2.

2 2Hydrocarbons of WasteOrganics ( ) Acetate + Organic Acids + CO + HAcetogens

2 2 4Organic Acids + CO H ( ) CH + Byproducts + EnergyMethanogens+

Anaerobic microbes are important for degrading the halogens (reductive dehalogenation)

and nitrosamine, reduction of epoxides to olefins, reduction of nitro groups and ring fission of aromatic structures. Several microbes have been identified which can break down the chlorinated substances like PCBs. Majority of the organochlorines appears to be biotransformed, forming cunjugates with the soil humic matter.

Advantages of Anaerobic Treatment Anaerobic treatment has several attributes that are considered favorable in biological

treatment- 1. Less energy is required as mechanical aeration is avoided. On the contrary a potential

energy source methane (CH4) is produced. 2. Significantly less land is required with smaller reactor volumes;


3. Extremely high destruction rates of wastes molecules can be achieved; 4. Solids (biological sludge) generation from the growth of biological cells (biomass

production) is about 20 times less than the aerobic process, and therefore cost of disposal of solids are reduced;

5. Certain toxic organic wastes can only be destroyed by anaerobic digestion; 6. Many industrial wastewater lacks sufficient growth nutrients to support aerobic

bacteria. Fewer nutrients are required to support the anaerobes; 7. There is complete elimination of off-gas air pollution.

Disadvantages 1. Longer start-up time. While it is in days for aerobic process, it may be in months for

anaerobic process; 2. May be more susceptible to upsets due to toxic substances; 3. It has potential for production of odors and corrosive gases; 4. It is not possible to remove biological nitrogen (N) and phosphorus (P); 5. May require alkalinity addition.

Anaerobic Digestion Anaerobic digestion is a process of oxidizing chemically contaminated materials in

closed vessels in the absence of air.

(b). Aerobic Degradation Aerobic process requires adequate supply of molecular oxygen to decompose the organic

matters and retrieve energy from it, which is needed by the bacteria to grow and multiply. The aerobic biodegradation process is rapid and more complete and there is no problematic end products like methane (CH4) and hydrogen sulfide. Naturally occurring aerobic bacteria can decompose both natural and the synthetic hazardous organic materials to harmless CO2 and water.

2 2Organics + Molecular Oxygen (Aerobes) CO + H O + Other products + Energy

Many hazardous organics are readily degraded aerobically, but because the depth of

oxygen penetration in soils is limited, the rate and extent of biological detoxification is also limited. Mechanical aeration may sometimes be needed in biological treatment of landfill leachates and heavily contaminated wastewaters. The dissolved molecular oxygen concentration must be maintained at least at 2 pounds of oxygen per pound of solids destroyed. If the oxygen content is too low, portions of the solids may become anaerobic and adversely affect biological destruction.


Microbial Destruction of Hazardous Wastes Microremediation technology is now used to detoxify the hazardous chemical wastes

before dumping in the land-fills and closing it finally. It has also been proposed for application in-situ by injecting naturally occurring microbes, cultured bacteria, nutrients and oxygen into the soil column or groundwater to form zones of microbial activity. The chlorinated and non-chlorinated pollutants (hazardous wastes) are broken down into carbon dioxide and water and a bacterial biomass is generated. Microremediation is typically able to reduce up to 90% of some contaminants in field systems. The technology has been successfully used in Australia for treatment of PCBs at a site in NSW and for cleaning up chlorinated phenols at a herbicide manufacturing plant near Melbourne. Animal manure were spread over the contaminated sites and in about an year full degradation of the contaminants occurred by mixed species of bacteria.

Plant Assisted Microbial Degradation Many organic compounds are degraded by microorganisms located in the rhizospheres

(in the root zone) of plants. Microbial activity in the rhizosphere of plants is several-fold higher than in the bulk soil. The population of microflora present in the rhizosphere is much higher than present in the vegetation-less soil. (Suthersan, 1997). Certain plant roots release substances that are nutrients for microorganisms, bacteria and fungi, that provide favorable habitats for soil microbes to act (Cunningham and Ow, 1996).

A typical microbial population present in the rhizosphere, per gram of air-dried soil comprises

1). Bacteria: 5 106 2). Actinomycetes: 9 105 3). Fungi: 2 103 This results in increased biological activity of the microbes in the area immediately

surrounding the root zone (rhizosphere). By encouraging a microbiologically active rhizosphere, the plants facilitate accelerated digestion (biodegradation) of wide variety of organic contaminants in the upper soil layers and/or wastewater/polluted water (Anderson et al. 1993). Interestingly, gram-negative bacteria appear to have some important metabolic capabilities for degrading xenobiotic chemicals not found in gram-positive bacteria.

A large number of bacteria and fungi are capable of degrading the hazardous chemical chlorobenzene and mineralizing it. The end products are 2 & 4 chlorophenol. Researches indicate that certain herbs stimulates microbial degradation of chlorinated organics primarily via rhizospheric reductive dechlorination biodegrdation processes. Chlorobenzene can undergo microbial dechlorination and the benzene ring can be converted to catechol, followed by ring fission or oxidation of the side chain.


Role of Environmental Biotechnology & Genetic Engineering In Improving Microbial Degradation by Producing Genetically Engineered Bacteria

With the advancement in our knowledge in environmental biotechnology some bacterium

have been genetically engineered (tailored) to biodegrade those hazardous wastes and chemicals which otherwise cannot be degraded by the naturally occurring bacteria. In Germany, genetically engineered bacteria have been used to clean up polluted soils and oil spills in water. These bacteria eat pollutants, gobble them up and chemically alter them in their bodies. When pollutants are finished the bacteria die leaving clean soil and a biomass of dead bacteria containing harmless minerals.

With genetic engineering it will be possible to enhance the reduction activities of hexavalent chromium {Cr (VI)} of the indigenous bacterial strains that express such activities at high levels under poor nutrient and stressful environmental conditions.

Development of Microbial Biosensors by Genetic Engineering Genetic engineering has led to development of microbial biosensors. These are

genetically engineered bacteria. They offer the potential to measure quickly, cheaply and accurately the degree of contamination of environmentally contaminated sites. More specifically, biosensors have been developed to detect heavy metals in contaminated sites. These microbial biosensors have used two distinct methods to detect heavy metal ions-

1). Protein (antibodies & enzymes) or 2). Whole cells (genetically modified or not)

Table 5. Degradable Organic Hazardous Wastes and the Associated Microorganisms

Hazardous Organic Compounds Microorganisms Involved in Degradation Phenylmercuric acetate Pseudomonas, Arthrobacter, Citrobacter, Vibrio Raw rubber, hevea latex Actinomycetes Detergents Nocardia,Pseudomonas PCBs Not identified Malathion Trichoderma, Pseudomonas Endrin Arthrobacter, Bacillus Lindane Closteridium DDT Escherichia, Hydrogenomonas, Saccharomyces Dieldrin Mucor

Sources: Various Publications of UNEP, WWF and WHO (1992-2002) Various biosensors have been designed to evaluate the heavy metal concentrations of

metals like cadmium (Cd), mercury (Hg), nickel (Ni), copper (Cu), arsenic (As) and iron (Fe). (D Souza, 2001; Verma & Singh, 2005; Bruschi & Goulhen, 2006).


Table 6. Genetically Engineered Bacteria Capable of Destroying Some Hazardous Chemicals

Bacterium Chemicals Destroyed 1. P. putida Camphor degradation 2. P. oleovarans Alkane degradation 3. P. cepacea 2,4,5 Trichlorophenoxyacetic acid degradation 4. P. mendocina Trichloroethylene degradation 5. P. diminuata Parathion (pesticide) degradation

Source: Biotechnology and Biodegradation ; Kamely et,al. (1990)

Factors Affecting Microbial Remediation Microbial remediation depends upon the presence of appropriate micro-organisms in the

correct amounts and in combinations and in appropriate environmental conditions. Bio-stimulation & bio-augmentation are two essential factors influencing bioremediation by microbes.

Bio-Stimulation It is the addition of nutrients (usually source of carbon, nitrogen & phosphorus), oxygen

or other electron donors or acceptors. These amendments serve to increase the number or activity or both, of naturally occurring micro-organisms available for bioremediation. Amendments can be added in either liquid or gaseous forms, via injection. Liquids can be injected into shallow or deep aquifers to stimulate the growth of micro-organisms involved in bioremediation. Anderson et al. (2003) removed uranium (U) from groundwater of an uranium-contaminated aquifer by biostimulating the in-situ activity of Geobacter spp..

Bio-augmentation

It is the addition of microorganisms that can bio-transform (usually toxic metals) or biodegrade (toxic organic compounds) a particular contaminant.

Success Story of Microbial Remediation for Environmental Cleanup in UK The town of Salem in New Hampshire, UK, faced a big environmental problem from the

decommissioned wastewater treatment plant in 1987 (constructed in 1964) which showed symptoms of soil and groundwater contamination by chlorinated solvents e.g. the chlorinated aliphatic hydrocarbon (CAH) at unacceptable levels. The potential impact of the CAHs could have been on the 30 bedrock water supply wells located within a 3 km radius of site. The major challenge for the cleanup was the large area (2-3 ha) of contaminated land and groundwater.

Bioremediation program was started in the year 2000 and included injections of 5 bio-stimulant (mixture of yeast & lactose) at the site. A total of more than 200,000 lb (90,720 kg) of bio-stimulant was injected during the program to enhance the anaerobic destruction of


chlorinated solvents in groundwater. Bioremediation successfully destroyed the chlorinated solvents mass at an expenditure of just US $ 300,000.

It saved the municipalities an estimated US $ 2 million in cleanup cost by conventional treatment approach (pump and treat) that would have required up to 20 extraction wells, a treatment building with air-stripper, treatment of the air stripper discharge gas, and at least 10 years of system operation and monitoring. (Schaffner, 2004).

The Limitations of the Microbial Technology A limitation of the use of microbial technology for metal remediation is that although the

metals are bound to microbes, they can be released back into the soil soon after decomposition of the microbes upon their death and decay and still be present in soil. The in-situ microbial remediation strategies may need to combine with phyto-remediation strategies by suitable hyper-accumulator plants that can effectively uptake the metals (made bioavailable by the microbes) from soil and bioaccumulate them in their roots & shoots, thus preventing their recycling in soil. Genetic engineering can resolve the problem by engineering bacteroids (with metal binding peptides) in root nodules of plants. This has been termed as symbiotic engineering.

II. PLANTS ASSISTED BIOREMEDIATION (PHYTOREMEDIATION) Phytoremediation (Greek: phyton = plant; Latin: remediare = remedy) is emerging green

bioengineering technology for environmental cleanup that uses plants to remove pollutants from the soil or to render them harmless. It takes advantage of the natural abilities of plants to take up, bioaccumulate, store or degrade organic or inorganic substances. They are cost-effective, aesthetically pleasing, passive, solar-energy driven and pollution abating natures biotechnology meeting the same objectives of fossil-fuel driven and polluting conventional technology. Plants involved in phytoremediation are adapted to thrive in very harsh environmental conditions of soil and water; absorb, tolerate, transfer, assimilate, degrade and stabilize highly toxic materials (heavy metals, radionuclides and organics such as solvents, crude oil, pesticides, explosives, chlorinated compounds and polyaromatic hydrocarbons) from the polluted soil and water. The organic pollutants may ideally be degraded to simpler compounds like carbon dioxide (CO2) and water (H2O), thus reducing the environmental toxicity significantly. Possibly due to their static (non-mobile) nature, plants had to evolve their survival modes even in odd environments including lands contaminated with xenobiotic substances.

Large scale phytoremediation of the contaminated sites has been achieved for heavy metals , organic xenobiotics, and radionuclides. Phytoremediation has been carried out commercially or has been demonstrated as pilot scale studies on nearly 200 contaminated sites (involving all categories of organic and inorganic chemical and radiological contaminants) in the U.S. The plant biomass eventually becomes valuable biological source for the community or for the plant based industries. The roots, shoots and leaves may be collected (harvested) and incinerated to decompose the contaminants.


Contaminants Suitable for Removal by Phytoremediation The following recalcitrant organic & inorganic pollutants & radioactive contaminants are

best suited for phytoremediation technology- 1. Benzene 2. Toluene 3. Ethylbenzene 4. Xylene 5. Chlorinated Solvents (TCE & PCE) 6. Chlorinated Pesticides 7. Organophosphates 8. Insecticides 9. Polyaromatic Hydrocarbons (PAHs) 10. Polychlorinated Biphenyls (PCBs) 11. Nitrotoluene ammunition wastes 12. Petroleum Hydrocarbons 13. Nutrients like nitrate, phosphate and ammonium 14. Toxic heavy metals (Cadmium, Lead, Nickel, Chromium, Mercury, Arsenic etc.) 15. Hazardous Air Pollutants e.g. Oxides of Sulfur & Nitrogen 16. Radionuclides (Cesium, Strontium, Uranium etc.)

Commercial Organizations Involved in Phytoremediation & Business Potential

In the last few years, several commercial companies practicing phytoremediation

biotechnology for environmental cleanup have come up in US and Europe. Important among them are Phytotech (USA), Phytoworks (USA), Earthcare (USA), Ecolotree (USA), BioPlanta (Germany), Piccoplant (Germany), Plantechno (Italy), Slater (UK) and Aquaphyte Remediation (Canada). A number of large industrial companies, mainly the oil and chemical industry, are also either employing phytoremediation technology or supporting it through appropriate funding.

Sources & State of Heavy Metals in Soil and their Bioavailability for Phytoremediation

Developmental activities and their byproducts are the sources of metal contamination of

our soils & water bodies. Main sources are 1). Metalliferous Mining & Smelting : Arsenic (As), cadmium (Cd), lead (Pb) &

mercury (Hg); 2). Industries (Metal & Electroplating, Saw Mills etc.). Chromium (Cr), cobalt (Co),

copper (Cu), Zinc (Zn), nickel (Ni), As, Cd & Hg;


3). Atmospheric Deposition from Industries & Automobiles: Uranium (U), As, Cd, Cr, Cu, Pb & Hg;

4). Agricultural activities: Selenium (Se), As, Cd, Cu, Cr, Pb, Zn & U 5). Waste disposal (MSW landfills & wastewater treatment plants): As, Cd, Cr, Cu, Pb,

Hg, & Zn 6). Wastewater from electroplating, paint and cement industries discharge heavy metals

like cadmium (Cd), copper (Cu), lead (Pb), mercury (Hg) nickel (Ni), zinc (Zn) and arsenic (As).

Heavy metals are defined as metals having density more than 5 g/cm3. The group of

heavy metals are about 65. Some heavy metals, such as cobalt (Co), chromium (Cr), copper (Cu), nickel (Ni) & zinc (Zn) are essential and serve as micronutrients for plants like calcium (Ca), potassium (K), magnesium (Mg), manganese (Mn), iron (Fe) and sodium (Na). They are used for redox-processes, as components of various enzymes and for regulation of osmotic pressure in cells. Other metals have no biological role e.g. cadmium (Cd), lead (Pb), mercury (Hg), aluminum (Al), gold (Au) and silver (Ag). Some of them e.g. Cd2+, Ag2+, Hg2+ tend to bind the SH groups of enzymes and inhibit their activity (Turpeinen, 2002).

Heavy metals are one very significant category of the industrial pollutants which are unique being selectively toxic, persistent and non-biodegradable. At high concentrations, both essential and non-essential metals can damage cell membrane, alter enzyme specificity, disrupt cellular function, and even damage the structure of DNA. They have been linked to birth defects, cancer, skin lesions, retardation leading to disabilities, kidney & liver damage and several other health problems. Remediation of metals presents a different set of problems when compared to organics. Organic compounds can be degraded while metals normally need to be physically removed by excavation or immobilized by plant roots.

Metal Concentrations in Soil & Their Bioavailability for Uptake by Plants Metal concentration in soil can range from < 1 mg / kg to as high as 100,000 mg / kg (Eapen

et al. 2006). Metals are often tightly bound to soil particles. Cations of heavy metals are often bound to soil particles because of soil cations exchange capacity. Binding mechanisms of heavy metals is complex and vary with composition of soil, soil acidity and redox conditions. The binding affinity of cations reduces cation movement in vascular plants, particularly in the negatively charged xylem cells. The slow desorption of heavy metals in soil has been a major impediment to the successful phytoremediation program of the metal contaminated sites.

Metal bioavailability is often low in soil systems. Metals are more bioavailable at acidic pH values. Generally, only a fraction of soil metal is readily available (bio-available) for the plant uptake. In the soil, the organic matter and the clay mineral content are important factors that can reduce metal bioavailability. Clays, with high cation exchange capacities, such as montmorillonite, appear to reduce metal bioavailability & toxicity. In a study used to investigate the effect of cadmium (Cd) on microbial biodegradation of toxic organic compound phenanthrene, a total of 394 mg cadmium (Cd) per kg of soil was added, but only 3 mg cadmium (Cd)/L were actually bioavailable. Similarly only 1 % of the total zinc (Zn) used in a study was present in the aqueous phase. In another study, 20 mg/L of soluble metal initially amended was below the detection limit i.e. 0.03-0.04 mg/L. At 100 mg/L of total


metal amended, only 1mg cadmium (Cd)/L, and less than 0.12 mg copper (Cu)/L and chromium (Cr)/L were found in the aqueous phase. (Sandrin & Hoffman, 2006). Plant roots also increase metal bioavailability by extruding protons to acidify the soil and mobilize the metals. (Zhao et al., 2001). By decreasing the pH below 5.5, metal bioavailability for plant roots can be enhanced. But that may also inhibit plant growth.

The Soluble & Mobile Metals The bulk of the metal in soil is commonly found as insoluble compounds unavailable

for transport into the plant roots from the aqueous phase. Metals, which are taken up by plants are those which exist as soluble components in the soil solution or are easily desorbed or solubilized by root exudates. Solubility of metals is dependent on soil characteristics and is strongly influenced by pH of the soil and the degree of complexation with soluble ligands.

However, some metals, such as zinc (Zn) and cadmium (Cd) are considered as easily mobile heavy metals as they occur primarily as soluble or exchangeable form and readily bioavailable. Copper (Cu), chromium (Cr) and molybdenum (Mo) are mainly bound in silicates and thus are slightly mobile and available. Others such as lead (Pb), occur as insoluble precipitate (phosphates, carbonate & hydroxy-oxides) in soil which is significantly much less mobile and largely unavailable for plant uptake.

Table 7. Phytoremediation Markets in the U.S. in 2005

Phytoremediation Works Carried Out Value in Million US Dollars 1. Removal of Heavy Metals from Contaminated Soil 70 100 2. Removal of Heavy Metals from Contaminated Groundwater 1 3 3. Removal of Heavy Metals from Wastewater 1 2 4. Removal of Radionuclides 40 80 5. Removal of Organics from Contaminated Groundwater 35 70 6. Others 65 115 Total 214 370 Million Dollars

Source: Eapen et al. (2006)

Table 8. Range of Heavy Metals in Contaminated Soil

Metals Contamination Range (g/kg) Regulatory Limit (mg/kg) Lead (Pb) 1000 6,900,000 600 Arsenic (As) 100 102,000 20 Cadmium (Cd) 100 - 345,000 100 Chromium (Cr) 5.1 3,950,000 100 Copper (Cu) 30 550,000 600 Mercury (Hg) 0.1 - 1,800,0000 270 Zinc (Zn) 150 5,000,000 1,500

Source: Eapen et, al; In SN Singh & RD Tripathi (ed.) Environmental Bioremediation Technologies ; Springer (2006).


Plant Species Involved in Phytoremediation Several plants are being identified and trialed to be used in phytoremediation task. The

most versatile plant species that has been identified after rigorous laboratory and field experiments are-

A number of them are still wild, while others have been domesticated for their food value. They are highly salt and toxicity tolerant, have extensive root binding system and were tried in the rehabilitation works. Number of them readily absorb, volatilise and/or metabolise compounds such as tetrachloroethane, trichloroethylene, metachlor, atrazine, nitrotoluenes, anilines, dioxins and various petroleum hydrocarbons. Ideal species for the job are members of the grass family Graminea and Cyperacea and the members of families Brassicaceae (in particular the genera Brassica, Alyssum and Thlaspi), and Salicaceae (in particular willow and poplar trees). Grasses such as the vetiver, clover and rye grass, Bermuda grass, tall fescue etc. have been particularly effective in the remediation of soils contaminated by heavy metals and crude oil (Kim, 1996).

Table 9.

Important Plant Species Identified for Phytoremediation Works 1. Sunflower (Helianthus anus); 2. Vetiver grass (Vetiveria zizanioides); 3. Indian Mustard (Brassica juncea) 4. Poplar tree (Populus Spp.); 5. Brake fern (Pteris vittata) 6. Barmuda grass (Cynodon dactylon); 7. Bahia grass (Paspalaum notatum); 8. Cumbungi (Typha angustifolia); 9. Redroot pigweed (Amaranthus retroflexus); 10. Kochia (Kochia scoparia); 11. Foxtail barley (Hordeum jubatum); 12. Switch grass (Panicum variegatum); 13. Musk thistle (Carduus nutans); 14. White raddish (Raphanus sativus); 15. Catnip (Nepeta cataria); 16. Big bluestem (Andropogan gerardii) 17. Alpine pennycress (Thlaspi Spp.); 18. Canada wild rye (Elymus candensis) 19. Nightshade (Solanum nigrum); 20. Wheat grass (Agropyron cristatum) 21. Alfa-alfa (Medicago sativa); 22. Tall Fescue (Festuca anundinacea) 23. Lambsquarters (Chenopodium berlandieri); 24. Reed grass (Phragmites australis); 25. Tall wheat grass (Thynopyron elongatum); 26. Rhodes grass (Chloris guyana); 27. Flatpea (Lathyrus sylvestris); 28. Carrot (Daucus carota) 29. Willows (Salix viminalis) 30. Periwinkle (Cathranthus roseus);


Table 10. Some Known Hyperaccumulator Species of Different Metals

Metals and Plant Species Concentration of Metal Accumulated (mg/kg)

A. Nickel (Ni) Thlaspi spp. (Brassicaceae) 2000 31,000 Alyssium spp. (Do) 1280 29,400 Berkheya codii (Asteraceae) 11,600 Pentacalia spp. (Do) 16,600 Psychotria corinota (Rubiaceae) 25,540 B. Zinc Thlaspi caerulescene (Brassicaceae) 43,710 Thlaspi rotundifolium (Do) 18,500 Dichopetalum gelonioides

(Do) 30,000

C. Cadmium Thlaspi caerulescene (Brassicaceae) 2,130 D. Lead Minuartia verna (Caryophyllaceae) 20,000 Agrostis tenius (Poaceae) 13,490 Vetiveria zizaniodes > 1500 E. Cobalt Crotolaria cobalticola (Fabaceae) 30,100 Haumaniastum robertii (Lamiaceae) 10,232 F. Copper Ipomea alpina (Convolvulaceae) 12,300 G. Manganese Maystenus sebertiana (Celastraceae) 22,500 Maystenus bureaviana (Celastraceae) 19,230 Macadania neurophylla (Proteaceae) 55,200 H. Selenium Astragalus racemosus (Leguminosae) 1,49,200 Lecithis ollaria (Lecithidiaceae) 18,200 I. Arsenic Pteris vittata (Fern)

Source: Eapen et, al; In SN Singh & RD Tripathi (ed.) Environmental Bioremediation Technologies ; Springer (2006). Hybrid poplar, willows, sunflower, alpine pennycress, clover, Indian mustard, redroot

pigweed and ferns have been plants of choice for many commercial phytoremediation applications.

The Hyper-Accumulator Species There are species which can bioaccumulate very high concentrations of metals in their

stem and leaves. These are hyper-accumulators species. More than 400 plant species ranging from herbs to trees have now been identified from the families of Euphorbiacea, Asteraceae, Brassicaceae and Rubiaceae which are hyperaccumulators of metals (Eapen et al.,


2006). Most have been found in the contaminated areas of temperate Europe and the USA, New Zealand and Australia. They can accumulate 100 - 500 times higher levels of metal concentrations in their above-ground parts. (Chaney et al. 1997). Plants are called hyperaccumulators when they can accumulate more than 0.1% Pb, Co, Cr or more than 1 % Mn, Ni, or Zn in plant shoots when grown in their natural habitats (Baker & Brooks, 1989). The degree of accumulation of metals like Ni, Zn, and Cu observed in hyperaccumulator species often reaches 1 5 % of their dry weight. (Raskin et al., 1997). Hyper-accumulator plants tends to be contaminant specific. No plant species has been found that demonstrate a wide spectrum of hyper-accumulation (Watanabe, 1997).

These hyper-accumulator plants possess genes that regulate the amount of metals taken up from the soil by roots and deposited other locations within the plants. There are number of sites in the plant that could be controlled by different genes contributing to the hyper-accumulation genetic traits. These genes govern processes that can increase the solubility of metals in the soil surrounding the roots as well as the transport proteins that move metals into the root cells.

It may be possible to revegetate closed hazardous landfills, industrial sites, and dis-used mine areas with hyper-accumulator species. Recent researches have indicated that the range of soil types suitable to hyper-accumulators can be widened by the incorporation of essential nutrients (e.g. N and P), and also chelating agents like EDTA which increase the plant availability of metals, particularly those in clay-textured soil.

Mechanism of Phytoremediation of Contaminated Soils The plants act as accumulators and excluders. Accumulators survive despite

concentrating contaminants in their aerial tissues. They biodegrade or biotransform the contaminants into harmless forms in their tissues. Adaptations to tolerate toxic compounds in plants appears to be processes like immobilization, exclusion, chelation & compartmentalization. These mechanisms not only control the uptake and accumulation of essential and non-essential heavy metals, but also detoxify them.

Phytoremediation of organic and inorganic contaminants involves either physical removal of compounds (phyto-extraction) or their bioconversion (phyto-degradation or phyto-transformation) into biologically inert forms. The conversion of metals into inert forms can be enhanced by raising the pH (e.g. through liming), or by addition of organic matter (e.g. sewage sludge, compost etc.), inorganic anions (e.g. phosphates) and metallic oxides and hydroxides (e.g. iron oxides). The plants themselves can play a role here by altering soil redox conditions and releasing anions and /or lignins (Salt et al. 1995).

Phytoremediation technology works mainly through

1). Phytoextraction (Phytoaccumulation of Contaminants) Plant roots uptake (extract) metal or radioactive contaminants from the soil, polluted

water and the wastewater, translocate and accumulate them in their roots. Plant roots absorb both organics and inorganics. Considerable amount of the contaminants may be translocated above through the xylem and accumulated in the shoots and leaves. The radionuclide uptake by plant roots need not necessarily result in translocation to shoots. The majority of cesium-


137 (Cs137) taken up by plants tends to be localized in roots. However, 80 % of the strontium-90 (Sr90) taken up by the plants, is usually localized in the shoots. (Eapen et al., 2006).

Phytoextraction exploits vascular plants natural ability to take up variety of chemical elements (macro & micronutrients) through the root system, deliver them to the vascular tissues, and to transport and compartmentalize in different organs.

Major limiting factor for phytoextraction are lower availability of metals in soil and poor translocation from roots to shoots. The bioavailability of a given compound for phytoextraction depends upon the lipophilicity and the soil or water conditions e.g. pH and clay content. Addition of soil amendments increased the metal availability in solutions to more than 10-fold for Cs137 and 100-fold for lead (Pb) and uranium (U). (Huang et al. 1998).

This technique has been effectively used by Phytotech Inc. (USA) for removal of lead (Pb) and cadmium (Cd) from contaminated soil. Excessive selenium (Se) in farm soils is also successfully remediated by this technology. (Eapen et al., 2006). Above-ground biomass, loaded with metals or radionuclides is harvested, processed for volume reduction and further element concentrations & safely recycled to reclaim metals of economic importance (industrial uses), or disposed off as hazardous waste in secured landfills or incinerated in case of radionuclides.

2). Phytostabilization (Immobilization of Contaminants)

Phytostabilization is stabilizing process for contaminated soils and sediments in place using plants, thus preventing the lateral or vertical migration of toxic metals by leachating. Certain plant species immobilize contaminants in the soil and groundwater through absorption by and adsorption on to roots or precipitation within the root zone (rhizosphere). Plants capable of tolerating high level of metal contaminants and having efficient growth rate with dense root systems (to bind/sorb contaminants) & canopies are preferred. Trees which transpire large amounts of water for hydraulic control (e.g. poplar) and grasses with fibrous roots (e.g. vetiver grass) to bind & hold soil are best suited for phytostabilization. Generally, plants suitable for soil conservation is good for phytostabilization of soil contaminants.

This technique is best applicable in phytostabilizing metal contaminants of waste landfill sites (e.g. Pb, Cd, Zn, As, Cu, Cr, Se, and U) where the best option is to immobilize them in situ. Addition of manure, digested sewage sludge, straw etc. to inorganic waste sites may help in binding of metals. Supplementing with limes (CaO) and limestones (CaCO3) may help neutralizing acid soils that help in binding of cationic metals with inorganic wastes.

Phytostablization is particularly suitable for stabilizing radionuclide-contaminated sites, where one of the best alternative is to hold contaminants in place to prevent secondary contamination and exposure to humans and animals. Plants roots also help to minimize water percolation through soil, thus reducing radionuclide leaching significantly. The technology is very suitable for controlling tailings in arsenic, zinc, cadmium and uranium mining areas.

Success Story

Mine tailings at Superfund site at South Dakota in the US containing up to 1000 mg/kg of arsenic (As) and lower concentrations of cadmium (Cd); smelter in Kansas, U.S, with up to 200,000 mg/kg of zinc (Zn) could be phytostabilized by decreasing vertical migration of leachate to groundwater using hybrid poplar (Populus spp.) trees (Hse, 1996).


Table 11. Plants Identified With Potential for Phytoextraction of Metals

Plant Species Metals Extracted 1. Indian mustard (Brassica juncea) Cd, Cr, Cu, Ni, Zn & Pb 2. Sunflower (Helianthus annus) Do 3. Rapeseed Do 4. Amaranthus (Amaranthus retroflexus) Do 5. Chenopodium (Chenopodium album) Do

3). Phytovolatilization (Evapo-transpiration of Detoxified Contaminants)

Phytovolatilization exploits a plants ability to transpire large amount of water from their leaf pores (stomata). Plants absorb and transpire the impurities from soil through their aerial organs (leaves). This system is a combination of hydraulic control and enhanced evapotranspiration.

Phytovolatilization is a mechanism by which plants convert a contaminant into volatile form and transpire the detoxified vapor through their aerial organs. Rate of transpiration is a key factor for this technology. For a 5 year old poplar tree (Populus Spp.) it was estimated to be 26 gpd (gallon per day) . A single willow tree (Salix Spp.) has been found to transpire 5000 gpd and an individual cottonwood tree transpire 50 to 35 gpd.

Some contaminants like selenium (Se), mercury (Hg) and volatile organic compounds (VOCs), can be released through the leaves into the atmosphere. (Cunningham and Ow, 1996). It is also being used for remediation of tritium (H3) contaminated water. Some transgenic plants e.g. Arabidopsis thaliana have been found to convert organic and inorganic mercury salts to the volatile and elemental form. (Watanabe, 1997).

4). Rhizofiltration

Rhizofiltration is the use of plant roots to sorb, concentrate or precipitate metal contaminants from aqueous medium. Although the technology is more suitable for decontamination of polluted water or removal of organic and inorganic impurities from wastewater by the use of aquatic plants, terrestrial species can also be used by growing hydroponically or on floating platforms in treatment ponds. An ideal plant for rhizofiltration should quickly produce significant amount of root biomass with large surface area when grown hydroponically.

5). Phytostimulation (Microbe Stimulated Phytoremediation)

Soil microbes have been found suitable to enhance the bioavailability of metals for phytoremediation and increase the phytoremediation potential of plants by complimenting the process in many ways. Soil microbes may degrade organic pollutants into simpler organic compounds and supply them as nutrient to plants for enhanced phytoremediation of the polluted site.

Microbial activity in the rhizosphere of plants is several-fold higher than in the bulk soil. The population of microflora present in the rhizosphere is much higher than present in the vegetation-less soil. It is due to the presence of nutrients from the root exudates of plants. (Suthersan, 1997).


A typical microbial population present in the rhizosphere, per gram of air-dried soil comprises bacteria 5 106, actinomycetes 9 105, and fungi 2 x 103 (Schnoor et al.,1995).

The rhizosphere in plants is divided into two general areas, the inner rhizosphere at the root surface and the outer rhizosphere immediately adjacent to soil. The microbial population is larger in the inner zone where the root exudates are concentrated. A large variety of exudates are secreted from roots in the form of sugars, amino acids and essential vitamins. Root exudates may also include acetates, esters and benzene derivatives. Enzymes are also present in the rhizosphere and this may act as substrates for the microbial population. Enzymes secreted by plant roots or the microbial community in the rhizosphere comprise esterases different oxido-reductases (phenoloxidases and peroxidases). Plant peroxidases are exuded by some members of Fabacea, Graminae and Solanaceae. White radish (Raphanus sativus) and horse radish (Armoracia rusticana) secrete peroxidase while the aquatic green algae Nitella & Chara secrete laccase. (Dias et al., 2006).

Chemolithotrophic bacteria have been shown to enhance metal availability. Several strains of Bacillus and Pseudomonas have been reported to increase cadmium accumulation by Brassica juncea. Naturally occurring Rhizobacteria were found to promote selenium (Se) and mercury (Hg) bio-accumulation in plants growing in wetlands. (Singh et. al., 2006).These microbes grow much better if organic manures are added to the soil.

Uptake of hydrophobic xenobiotics of larger size can be facilitated by primary microbial biodegradations in the rhizosphere. The hydrophobic persistent organic pollutants like polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with present log Kow value above 4 have been reported to be taken up by roots and transported to shoots in Cucurbita pepo. (Singh et. al., 2006).

Plants which have been identified to promote rhizosphere degradation of petroleum compounds are

1. Big bluestem (Andropogon gerardii), 2. Indian grass (Sorghastrum nutans), 3. Switch grass (Panicum virgatum), 4. Canada wild rye (Elymus canadensis), 5. Western wheat grass (Agropyron smithic) and some more. Aprill & Sims (1990) studied the degradation of 4 PAHs viz. benz(a)anthracene,

chrysene, benzo(a)pyrene and dibenz (a,h)anthracene in the root zone of mixture of 8 prairie grasses and found that the disappearance of the PAHs were consistently greater in the vegetated columns of soil than in the unvegetated controls.

Plants which have been identified to enhance microbial degradation of PAH compounds in the rhizosphere are

1. Alfalfa (Meticago sativa), 2. Fescue (Festuca anundinacea), 3. Big bluestem (Andropogon gerardii), and 4. Sudan grass (Sorghum vulgare sudanense). Wheat grass (Agropyron cristatum) has been found to enhance microbial degradation of

pentachlorophenol (PCP) and Phragmites australis the microbial degradation of phenols in


the soil. Mackova et al. (1997), reported effective degradation of PCBs (Polychlorinated Biphenyls) by cells of Solanum nigrum that were infected with bacterial strains of Agrobacterium tumefaciens and A. rhizogenes. Hairy root cultures of S. nigrum proved capable of transforming PCBs under controlled conditions. Plant cells proved capable of transforming PCBs even after growth had stopped. The red mulberry (Morus rubra) appears to be another good plant for enhancing microbial degradation of PCBs.

Root exudates in the form of compounds structurally analogous to the chemicals utilized by microorganisms, would enhance the activity of the bacteria present in the rhizosphere accelerating the degradation process. Certain phenolic compounds in the root exudates has been found to support growth of PCB-degrading bacteria and serve as structural analogs for PCB degradation. (Zhao et al., 2001).

Sometimes inoculation of plant soil by microbes enhance the phyto-degradation of organic contaminants present in the soil. Degradation of pentachlorophenol (PCP) was accelerated by the prosomillet after the soil in which it was growing was inoculated with Pseudomonas strain SR 3.

6). Mycorrhiza Assisted Phytoremediation

It is generally considered that the majority of plants growing under natural conditions have symbiotic relationship with mycorrhizal fungi roots, which results in increase in root surface area and enhanced absorption of nutrients. Mycorrhizal fungi have been reported in plants growing on heavy metal contaminated soils/sites indicating its heavy metal tolerance and a potential role in heavy metal phytoremediation. Mycorrhizal species Glomus, Gigaspora and Entrophospora, have been reported to be associated with most of the plants growing in the heavy metal polluted soils indicating its obvious role in assisting the plants in tolerating and removal of metals by phytoremediation. (Khan et. al.,2000).

Cellular Mechanism of Uptake & Detoxification of Heavy Metals by Plants: Role of Metal Chelators & Transporter Proteins

Chelation of heavy metals in the cytosol by high affinity ligands followed by subsequent

compartmentalization of the ligand-metal complex is thought to be a potentially very important mechanism of heavy metal detoxification and tolerance by plants. It is a process in which the metal cations binds to a chemical compound, resulting in an uncharged metal complexes that become easily bioavailable for uptake & transport in plants. Several natural and synthetic chelators are known. Among the natural chelators are organic acids, amino acids, phytochelatins (PC) and metallothionein (MT) and among the synthetic ones are ethylene diamine tetra acetic acid (ETDA) & ethylene glycol tetra acetic acid (EGTA). The use of natural & synthetic chelates has been shown to dramatically stimulate lead (Pb) accumulation in plants. They prevent Pb precipitation and keep the metal as soluble chelate - Pb complex bioavailable for uptake by plant roots & its transport to shoots. (Sriprang, 2006). Addition of synthetic chelates like EDTA has been found to be very effective in facilitating the plant uptake of Cd, Cu, Zn & Ni.(Raskin et al. 1997). Malic & Citric acids are two organic acids in plant roots that have been shown to make uncharged complexes with heavy metals.


Uptake and Translocation of Metals from Soil to Roots and to the Shoots : Role of Transporter Proteins

Accumulator plants must have the ability to uptake metals from the soil to roots and

translocate them to the shoots at high rates. Because of their charge, metal ions cannot move freely across the cellular membrane, which are lipophilic structures. Membrane proteins mediate ion transport into cells and they are known as transporter proteins (TP). They contain binding domains, which binds to specific ions from extracellular space through the hydrophobic environment of the membrane into cell. (Sriprang & Murooka, 2006). Transporter proteins are usually specific and one TP usually binds with an individual heavy metal only. Thus, Zinc (Zn) transporter protein (ZnTP1) can only uptake and bind with Zn but not with Cd.

Storage of Metals in Plant Cells : The Vacuolar Compartmentalization for Tolerance

The vacuole in plant cells appears to be the main storage site for metals.

Compartmentalization of metals in the vacuole appears to be the part of the tolerance mechanism of some metal hyper-accumulator plants. The nickel (Ni) hyper-accumulator T. goesingens enhances its Ni tolerance by compartmentalizing most of the intracellular leaf nickel components into the vacuole. (Sriprang & Murooka, 2006).

Role of Phytochelatins (PCs) PCs are metal-binding peptides in plants made of three amino acids (Glu, Cys & Gly) and

are know to play an essential role in the heavy metal detoxification by chelating heavy metals in the cytosol & sequestering PC- metal complexes in the plant cell vacuoles via transport across the tonoplast. Production of PC is activated/stimulated by heavy metals and the best activator is cadmium (Cd) followed by Ag, Bi, Pb, Zn, Cu, Hg and Au cation. It has been studied that Cd stimulates synthesis of PCs, which rapidly form a low molecular weight Cd-PC complex. The Cd-PC complex is transported into the vacuole by a Cd/H antiport and an ATP dependent PC-transporter protein. A gene (Hmt1), which codes for PC-transporter protein in yeast was isolated. The Hmt1 gene encodes for a member of family of ATP-binding cassette (ABC) membrane transport proteins that is located in the vacuolar membrane. The gene (Hmt1) product i.e. a protein (Hmt1) is responsible for transporting the Cd-PC complex into the vacuole. (Sriprang & Murooka, 2006).

PCs contain strongly nucleophilic sulfydryl group and thus can react with many toxic specie within the plant cell such as the free radicals, active oxygen species, and cytotoxic electrophilic organic xenobiotics and obviously heavy metals.


Role of Metallothioneins (MTs) MTs are super cysteine-rich metal-chelating proteins in plants meant to sequester toxic

heavy metals such as cadmium (Cd). MTs bind readily with Zn, Hg, Cu, Cd & Ag.

Fate of Chemicals Absorbed in Plant Roots & Shoot Cells Contaminants once absorbed inside the plant roots are either degraded into simple and

harmless compounds or transformed into some inert harmless materials. This they do with the aid of enzymes (described as biological catalysts) or symbiotic microbes inhabiting in their roots.

Phytodegradation Certain plant species breakdown the contaminants after absorbing them. This they do

through enzyme-catalyzed metabolic process within their root or shoot cells. Others breakdown the contaminants in the substrate itself by secreting enzymes and chemical compounds. The enzymes secreted are usually dehydrogenases, dehalogenases, oxygenases peroxidases, phosphatases, laccases and reductases. Plants can also degrade aromatic rings in complex organics with the aid of enzymes. Phenols have been found to be degraded by radish (Raphanus sativus) and potato (Solanum tuberosum) that contain peroxidase enzymes.

The biodegraded constituents are converted into insoluble and inert materials that are stored in the lignin or released as exudates (Watanabe, 1997). Enzymes cytochrome P450, peroxygenases & peroxidases are involved in plant oxidation of xenobiotics. Other enzymes like Glutathione s-transferase, carbo-oxylesterases, o-glucosyltransferases and o-malonyltransferases are associated with xenobiotic metabolism in plant cells & transportation of intermediates.

Several plants biodegrade contaminants with the aid of microbes which live in symbiotic association on their roots.

Phytotransformation Several inorganic and organic contaminants once absorbed inside the root, may become

biochemically bound to cellular tissues (biotransformed), in forms that are biologically inert or less active (Watanabe, 1997). In many cases, plants have the ability to metabolize organic pollutants by phytotransformation and conjugation reactions followed by compartmentalizing products in their tissues. Poplar trees (Populus Spp. have been found to transform trichloroethylene in soil and groundwater.


Table 12. Plant Enzymes Implicated in Phytodegradation & Phytotransformation of Organics & Inorganic Compounds

Enzymes Contaminants Degraded/Transformed into Less Toxic Forms 1). Phosphatase Organophosphates 2). Aromic Dehalogenase Chlorinated aromatic compounds (e.g. DDT, PCBs etc.) 3). O-demythlase Metaoalchor, Alachlor 4). Cytochrome 450, Peroxidases & Peroxygenases

PCBs

5). Glutathione s-transferase, carbo-oxylesterases o-glucosyltransferases, o-malonyltransferases

Xenobiotics

6). -cyanoanaline synthase Cyanide Sources : (Sandermann, 1994; Macek et al., 2000; Prasad, 2006).

Phytodetoxification of Toxic Cyanide (CN) by Phytotransformation Plants possess the necessary mechanism to detoxify the cyanides from mining wastes

resulting from gold & silver mining. Cyanide (ammonium thiocyanate) is the leach reagent of choice for gold (Au) and silver (Ag) extraction. During several metabolic functions, plants are confronted with cyanide as a byproduct of metabolism. This occurs particularly during synthesis of ethylene in mature tissues, where hydrogen cyanide (HCN) is formed as byproduct.

Consequently vascular plants have evolved effective strategies for detoxifying the toxic cyanide with the aid of enzymes. Those identified with cyanide detoxification are Salix spp. & Sorghum spp. Plants only survive cyanide exposure up to the doses they can eliminate. The cyanide detoxifying enzyme system is beta-cyanoalanine synthase which connects free cyanide and cysteine to cyanoalanine. The final metabolic product is aspargine, a non-toxic essential amino acids in plants.(Manning, 1988; Trapp et al., 2003).

Summary of the Metal Uptake & Transport in Plants 1. Metal ions are mobilized by secretion of chelators in soil and by acidification of the

rhizosphere (soil in the vicinity of root system); 2. Uptake of hydrated metal ions or metal-chelator complexes mediated by the

transporter proteins residing in the plasma membrane & energized by ATP (cellular energy molecules- adenosine triphosphate produced by the cell mitochondria);

3. Metals are chelated again in the cell cytosol by various binding natural chelators and ligands (PCs & MTs);

4. PCs quickly form complex with Cd (because cadmium is the best activator of PCs and PCs have greater affinity for Cd) and the PC-Cd complex is transported into the cell vacuole. Other metals also follow the same process and are transported into the cell vacuole.


SOME WONDER PLANTS FOR PHYTOREMEDIATION OF CONTAMINATED LANDS: POTENTIAL FOR COMMERCIALIZATION

1). The Vetiver Grass (Vetiveria zizanioides) The wonder grass vetiver is native of India, and has been used worldwide for

phytoremediation. It grows very rapidly and becomes effective for environmental restoration works in only 4-5 months as compared to 2-3 years taken by trees and shrubs for the same job. It can tolerate very high acidity and alkalinity conditions (pH from 3.0 to 10.5); high soil salinity (EC = 8 dScm), sodicity (ESP = 33 %) and magnesium; very high levels of heavy metals Al, Mn, Mg, As, Cd, Cr, Ni, Cu, Pb, Hg, Se, Zn and the herbicides and pesticides in soils.

Vetiver roots can absorb and accumulate several times of some of the heavy metals present in the soil and water. (Truong and Baker, 1998). Studies further indicated that very little (1 to 5 %) of the arsenic (As), cadmium (Cd), chromium (Cr) and mercury (Hg) and very moderate amount (16 to 33 %) of copper (Cu), lead (Pb), nickel (Ni) and selenium (Se) absorbed were translocated to the shoots above. Hence its green shoots can be safely used as feed or grazed by animals or harvested for mulch. Vetiver can be disposed off safely elsewhere, thus gradually reducing the contamination levels. Vetiver also has high capacity to absorb and remove agro-chemicals like carbofuran, monocrotophos and anachlor from soil thus preventing them from contaminating and accumulating in the crop plants.

Success Story

At the Scott Lumber Company site in Missouri, U.S., 16,000 tonnes of soils contaminated with polyaromatic hydrocarbons (PAHs) were biologically treated with VGT. The PAH concentration was effectively reduced by 70 %.(Pinthong et al., 1998).

2). The Brake Fern (Pteris vittata) It is hardy, versatile and fast-growing perennial plant easy to propagate. It can grow on

contaminated soils up 1,500 ppm of arsenic and is extremely efficient in extracting arsenic (As) from both soil and water and translocating it into its above-ground biomass mainly the fronds. It can reduce the concentration of 200 ppm of arsenic in water to nearly 100-fold (10 ppm) within 24 hours and can hold up to 22 grams of arsenic per kg of plant matter.

Brake fern has great potential to remediate arsenic contaminated soils and is very cost-effective. Arsenic concentration in fern fronds growing in soil spiked with 1,500 ppm arsenic increased from 29.4 ppm to 15,861 ppm in two weeks. In the same period, ferns growing on soil containing just 6 ppm arsenic accumulated 755 of arsenic in their fronds, a 126-fold enrichment. Arsen

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